DEVELOPMENT AND ANALYSIS OF MURINE MODELS FOR SICKLE CELL ANEMIA. The long term objective of this proposal is to continue the development of transgenic mouse models for sickle cell anemia and to use these models to study in vivo the pathogenesis of this disorder. During the prior funding period of this proposal, human sickling globin transgenes were introduced into two mutant murine backgrounds (beta-thalassemic and a newly developed strain of mice with a high oxygen affinity murine hemoglobin) in order to favor the in vivo deoxygenation and polymerization of the human sickling hemoglobins in murine red cells. The resulting animals exhibited a phenotype similar to that of individuals with sickle trait but not with sickle cell disease. As such the present proposal's principal objective is the construction of murine models for sickle cell anemia that mimic severe sickle cell disease. The methods we will use to genetically engineer mice so that their red cells contain exclusively or almost exclusively human sickling hemoglobin, will include: 1) homologous recombination in embryonic stem cells to inactivate the murine globins genes, 2) introduction of genes that code for murine globin antisense RNA, to decrease the expression of the murine globin genes, and 3) introduction of high expression human sickle globin constructs to direct the high level expression of the human globin chains in murine red cells. With these animals and with the previously developed murine models for sickle cell anemia, we plan to investigate in vivo; 1) the effect that a small amount of HbF and HbA have on murine sickle red cells, 2) the origins of different density populations of sickle red cells and the survival and primarily human sickling hemoglobins have on transgenic mice.